Overview of Mass Spectrometry for Protein Analysis

Mass spectrometry (MS) measures the mass-to-charge ratio of ions to identify and quantify molecules in simple and complex mixtures. MS has become invaluable across a broad range of fields and applications, including proteomics. The development of high-throughput and quantitative MS proteomics workflows within the last two decades has expanded the scope of what we know about protein structure, function, modification and global protein dynamics. This overview outlines the role of mass spectrometry in the field of proteomics, reviews MS methodology and instrumentation, and touches on sample preparation and liquid chromatography-based separation prior to MS analysis.

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Introduction to Mass Spectrometry

Mass Spectrometry Applications

Quantitative Proteomics

Sample Preparation

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Introduction to Mass Spectrometry

Proteomics is the study of all proteins in a biological system (e.g., cells, tissue, organism) during specific biological events (1, 2). The complementation of genomics and proteomics is considerably more difficult to study than genomics or even transcriptomics alone because of the dynamic nature of protein expression. Additionally, the majority of proteins undergo some form of post-translational modification (PTM), further increasing proteomic complexity. The broad scope of proteomics has only begun to be realized within the last 15 years due in large part to technological developments in mass spectrometry.

Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass and charge (m/z) ratio. Originally developed almost 100 years ago to measure elemental atomic weights and the natural abundance of specific isotopes (3), MS was first used in the biological sciences to trace heavy isotopes through biological systems; later on, MS was used to sequence oligonucleotides and peptides and analyze nucleotide structure (4).

The development of macromolecule ionization methods, including electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), enabled the study of protein structure by MS (5, 6, 7). This allowed scientists to obtain protein mass "fingerprints" that could be matched to proteins and peptides in databases to predict the identity of unknown proteins. Methods of isotopic tagging have led to the quantitation of target proteins both in relative and absolute quantities. Technological advancements have resulted in methods that analyze samples in solid, liquid or gas states. The sensitivity of current mass spectrometers allows one to detect analytes at concentrations in the attomolar range (10-18) (10).

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Mass Spectrometry Applications

Mass spectrometry (MS) measures the m/z ratio of ions to identify and quantify molecules in simple and complex mixtures. MS has become invaluable across a broad range of fields and applications, including proteomics. The development of high-throughput and quantitative MS proteomics workflows within the last two decades has expanded the scope of what we know about protein structure, function and modification, as well as global protein dynamics.

This overview outlines the role of mass spectrometry in the field of proteomics and reviews MS methodology and instrumentation. It also touches on sample preparation and liquid chromatography-based separation prior to MS analysis.

All mass spectrometers have an ion source, a mass analyzer and an ion detector. The nature of these components varies based on the type of mass spectrometer, the type of data required, and the physical properties of the sample. Samples are loaded into the mass spectrometer in liquid, gas or dried form and then vaporized and ionized by the ion source (e.g., APCI, DART, EI).

Schematic of the basic components of a mass spectrometer.

The charge that these molecules receive allows the mass spectrometer to accelerate the ions throughout the remainder of the system. The ions encounter electric and/or magnetic fields from mass analyzers, which deflect the paths of individual ions based on their mass and charge (m/z). Commonly used mass analyzers include time-of-flight [TOF], orbitraps, quadrupoles and ion traps, and each type has specific characteristics. Mass analyzers can be used to separate all analytes in a sample for global analysis, or they can be used like a filter to deflect only specific ions towards the detector.

Ions that have successfully been deflected by the mass analyzers then hit the ion detector. Most often, these detectors are electron multipliers or microchannel plates, which emit a cascade of electrons when each ion hits the detector plate (4). This cascade results in amplification of each ion hit for improved sensitivity. This entire process is performed under an extreme vacuum (10-6 to 10-8 torr) (8) to remove gas molecules, neutrals, and contaminating non-sample ions, which can collide with sample ions and alter their paths or produce non-specific reaction products (9).

Newer orbitrap technology captures ions around a central spindle electrode and then analyzes their m/z values as they move across the spindle with different harmonic oscillation frequencies. Orbitrap technology can achieve extremely high sensitivity and resolution of obtained mass spectra.

Mass spectrometers are connected to computers with software that analyzes the ion detector data and produces graphs that organize the detected ions by their individual m/z and relative abundance. These ions can then be processed through databases to predict the identity of the molecule based on the m/z.

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Diagram of a sector† mass spectrometer. A sample is injected into the mass spectrometer, and the molecules are ionized and accelerated. The ions are then separated by mass and charge by the mass analyzer via electromagnetic deflection, and the ions that are properly aligned are detected and amplified. The entire system is under intense vacuum during the entire process. After signal amplification, the data that is generated reports on the relative abundance of each ion based on its mass-to-charge (m/z) ratio. †Although sector instruments have decreased in use due to improvements in mass analyzers (e.g., quadrupole, orbitrap), this simplified diagram conveys a key principle of mass spectrometry, which is its ability to select and analyze specific ions in a complex sample.

Tandem mass spectrometry (MS/MS) offers additional information about specific ions. In this approach, distinct ions of interest are selected based on their m/z from the first round of MS and are fragmented by a number of methods of dissociation. One such method involves colliding the ions with a stream of inert gas, which is known as collision-induced dissociation (CID) or higher energy collision dissociation (HCD). Other methods of ion fragmentation include electron-transfer dissociation (ETD) and electron-capture dissociation (ECD).

These fragments are then separated based on their individual m/z ratios in a second round of MS. MS/MS (i.e., tandem mass spectrometry) is commonly used to sequence proteins and oligonucleotides because the fragments can be used to match predicted peptide or nucleic acid sequences, respectively, that are found in databases such as IPI, RefSeq and UniProtKB/Swiss-Prot. These sequence fragments can then be organized in silico into full-length sequence predictions.

Diagram of tandem mass spectrometry (MS/MS). A sample is injected into the mass spectrometer, ionized, accelerated and analyzed by mass spectrometry (MS1). Ions from the MS1 spectra are then selectively fragmented and analyzed by a second stage of mass spectrometry (MS2) to generate the spectra for the ion fragments. While the diagram indicates separate mass analyzers (MS1 and MS2), some instruments utilize a single mass analyzer for both rounds of MS.

Biological samples are often quite complex and contain molecules that can mask the detection of the target molecule, such as when the sample exhibits a large dynamic concentration range between the target analyte(s) and other molecules in the sample. Two methods of separation are commonly used to partition the target analyte(s) from the other molecules in a sample.

Gas chromatography (GC) and liquid chromatography (LC) are common methods of pre-MS separation that are used when analyzing complex gas or liquid samples by MS, respectively. LC-MS is typically applied to the analysis of thermally unstable and nonvolatile molecules (e.g., sensitive biological fluids), while GC-MS is used for the analysis of volatile compounds such as petrochemicals. LC-MS and GC-MS also use different methods for ionization of the compound as it is introduced into the mass spectrometer. With LC-MS, electrospray ionization (EI) is commonly applied, resulting in the production of aerosolized ions. With GC-MS, the sample may be ionized directly or indirectlyvia EI.

High performance liquid chromatography (HPLC) is the most common separation method to study biological samples by MS or MS/MS (termed LC-MS or LC-MS/MS, respectively), because the majority of biological samples are liquid and nonvolatile. LC columns have small diameters (e.g., 75 μm; nanoHPLC) and low flow rates (e.g., 200 nL/min), which are ideal for minute samples (2). Additionally, "in-line" liquid chromatography (LC linked directly to MS) provides a high-throughput approach to sample analysis, enabling multiple analytes to elute through the column at different rates to be immediately analyzed by MS. For example, 1-5 peptides in a complex biological mixture can be sequenced per second by in-line LC-MS/MS (2).

Quantitative Proteomics

While mass spectrometry can detect very low analyte concentrations in complex mixtures, MS is not inherently quantitative because of the considerable loss of peptides and ions during analysis. Therefore, peptide labels or standards are concomitantly analyzed with the sample and act as a reference point for both relative or absolute analyte quantitation, respectively. Commercial products are now available that allow the detection and quantitation of multiple proteins in a single reaction, demonstrating the high-throughput and global analytical platform that MS has become in the field of proteomics.

Relative quantitation strategies include stable isotope labeling using amino acids in cell culture (SILAC) and tandem mass tagging (TMT). In these approaches, proteins or peptides are labeled with stable isotopes that give them distinct mass shifts over unlabeled analytes. This mass difference can be detected by MS and provides a ratio of unlabeled to labeled analyte levels. These approaches are often used in discovery proteomics, where many proteins are identified across a broad dynamic range using different-sized labels.

Absolute quantitation is performed in targeted proteomic experiments and increases the sensitivity of detection for a limited number of target analytes. These approaches require spiking a sample with known amounts of synthetic peptides containing heavy stable isotopes, which act as internal quantitative standards for absolute quantitation of the corresponding natural peptides in the sample.

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Sample Preparation

All samples require some form of preparation prior to study by MS to remove detergents and to reduce the complexity of the sample when focusing on specific proteins and/or tag proteins for identification/quantitation. Proper sample preparation is critical for MS analysis, because the quality and reproducibility of sample extraction and preparation significantly impact results from MS instruments. Sample preparation encompasses a wide range of techniques that includes lysate preparation, protein or peptide enrichment, and sample clean-up and protein digestion. These techniques are described in detail in the accompanying article on sample preparation.